Project Summary/Abstract Mechanosensitive (MS) channels are oligomeric membrane proteins that function as mechano-electrical sensory switches in a wide range physiological processes. These include touch, hearing, proprioception, turgor control in plant cells and osmoregulation in bacteria. Among these, a fundamental class of MS channels responds to changes in the physical properties of the lipid bilayer by undergoing major structural transitions in response to membrane tension, thus fulfilling a major role in the response of living organisms to mechanical stimuli. This has been referred to as the ?force from lipid? principle of mechanosensitivity. The overall, long-term goal of this project is to understand the molecular mechanism of ?force from lipid? gating in mechanosensitive channels. Specifically, we will focus on the MscS family of MS channels found in most prokaryotes and plants. These channels are of fundamental importance in various physiological events, can been engineered for biomedical applications, and display fascinating intramembrane heterogeneity among family orthologs. More importantly, the MscS family Affords us the possibility of studying the functional behavior, high resolution structure and dynamics in the same MS system. Although MscL and MscS channels have been studied extensively and crystal structures have been available in multiple conformations, there are still major mechanistic questions that remain to be solved. This is particularly true for the molecular events underlying channel gating, in light of exciting new preliminary data at the core of this proposal. In this respect, we plan to experimentally address several fundamental questions: What is the physical basis of the energy transduction steps, starting with trans-bilayer tension and culminating in protein motion? What are the structures of the key functional states in its native, bilayer-embedded form? Where in the molecule does mechanical transduction occur? And how? Functional studies will be designed to understand the physical basis of energy transduction. Information on the architecture, dynamics and energetic relationship of MscS (plus other related members of the superfamily) with its surrounding lipid bilayer will be obtained from cryo-EM, EPR analysis of spin labeled mutants and computational methods. The data will be interpreted to generate high resolution structures of the different stages of the gating pathway in each type of channel. We suggest that the advent of new cryo-EM approaches to the analysis of structure and dynamics in membrane proteins in their native environment shall open an exciting new experimental avenue that will contribute to the understanding of biologically important events such as ion channel gating, nociception and signal transduction.